Carbohydrate polymers are the most pervasive organic compounds on earth. They function in many diverse biological roles, from serving as important structural elements to acting as signals. One important carbohydrate polymer is the arabinogalactan of mycobacteria. This cell wall glycoconjugate is composed of galactofuranose (Galf) and arabinofuranose (Araf) residues. The mycobacterial galactan is a polymer of 30-40 Galf residues, which are linked by alternating ?-(1->5), ?-(1->6) glycosidic linkages. An initial sequence of up to two Galf residues is assembled by the galactofuranosyltransferase GlfT1. The majority of the galactan is assembled by the galactofuranosyltransferase GlfT2. While the biosynthetic pathway for galactan formation is generally understood, the molecular basis for galactan length control is unclear. In mycobacteria, galactan assembly takes place on an initiator sequence consisting of the disaccharide rhamnosyl (Rha)-?-(1->3)-N-acetylglucosamine (GlcNAc) pyrophosphate linked to a C50 (decaprenyl) lipid. GlfT1 adds a sequence of up to two Galf residues, though the precise product it generates is unclear. The polymerase GlfT2 then adds 30-40 Galf residues to form the full-length galactan. The activity of GlfT1 has only been observed in cell extracts, and a clear substrate has not been described. Our laboratory has examined GlfT2 polymerization with synthetic substrates. While these synthetic substrates have provided insight into the mechanisms that underlie galactan assembly, they are missing key features present in the natural substrate. Polymerization by GlfT2 is directly affected by the length of the carrier lipid on the acceptor substrate. We will explore this effect with analogs of the natural substrate bearing different isoprene carrier lipids I propose to investigate the mechanism of galactan formation using substrates similar to those generated by the cell. I envision accessing these substrates by the chemoenzymatic relay synthesis of substrates for the rhamnosyltransferase WbbL and the galactofuranosyltransferases GlfT1 and GlfT2. Acceptor substrates for GlfT1 will be generated by a chemoenzymatic approach. Addition of L-Rha to synthetic acceptors by WbbL will provide the desired GlfT1 acceptor substrates. We will investigate the function of GlfT1 by analyzing the number of Galf additions it catalyzes. These studies will clarify the role of GlfT1 in galactan assembly. We shall then probe GlfT2 polymerization with the products of GlfT1 catalysis.